Project A: Serotonin mediates the potent inhibitory action of cocaine on synaptic transmission in the ventral pallidum Cocaine actions in the brain are largely thought to be mediated by blocking the dopamine transporter and enhancing dopamine concentration in the nucleus accumbens (NAc). However, non-dopaminergic actions of cocaine have been suggested to play a role in cocaine reinforcement and reward. This study examines cocaine action in the ventral pallidum specifically for the modulation of GABA transmission from direct and indirect pathway medium spiny neurons (dMSNs and iMSNs) of the NAc to neurons in the ventral pallidum. Our results show that cocaine selectively suppressed synaptic transmission from iMSNs to ventral pallidum neurons while it had no effect on transmission from dMSNs. Cocaine mediated inhibition was insensitive to the application of D2 receptor antagonist and was still present in D2 receptor knockout mice. Cocaine effect was mimicked by a specific serotonin transporter blocker citalopram, but not by the dopamine or noradrenaline transporter blockers. Serotonin (5-hydroxytryptamine, 5-HT) and a selective agonist for the 5-HT 1B/D receptor sumatriptan inhibited GABA transmission from iMSN to ventral pallidum neurons in pathway selective manner. Cocaine and 5-HT inhibition of GABA transmission was lost in 5-HT 1B receptor knockout mice. Lastly, voltammetry recordings of dopamine revealed that dopamine transient by optogenetic stimulation of midbrain projections to ventral pallidum was undetectable, yet the stimulation of the same projections in the NAc elevate dopamine level. Moreover, voltammetry recording of 5-HT indicated the evoked 5-HT transients in the ventral pallidum upon optogenetic stimulation of dorsal raphe projections. Taken together, these findings suggest that cocaine targets the 5-HT transporter in the ventral pallidum to elevate 5-HT concentration and selectively suppress synaptic transmission from indirect pathway neurons to the ventral pallidum via 5HT-1B receptors. This study reveals selective, pathway-specific actions of cocaine beyond the striatum that are mediated by 5-HT and independent of dopamine and its receptors. Project B: Role for dopamine D2 receptors in the stimulatory and depressive effects of alcohol Alcohol produces both stimulant and sedating effects in humans. Clinical literature suggests that people who experience the stimulant effects of alcohol more intensely are more likely to abuse the substance and develop dependency. In animal studies, stimulant and depressive effects of alcohol can be quantified by locomotive activity. Previous research indicates a role for dopamine receptors in the locomotive effects of alcohol, however, a detailed understanding of the receptor class and localization is unknown. The current study examines the role of specific subpopulations of dopamine D2 receptors in modulating the stimulant and depressant effects of alcohol. Using genetically engineered mice lacking D2 receptors on medium spiny neurons (MSNs) in the striatum or D2 autoreceptors on midbrain dopamine neurons, we examined dose-dependent alcohol-induced locomotion. Compared with littermate controls, MSN D2 knockout mice show a significantly increased locomotor response to alcohol, while the mice lacking D2 autoreceptors are more sensitive to the depressive effects of alcohol. To assess differences in alcohol-induced sedation in each of the mouse lines, we performed the Loss of Righting Reflex (LORR). LORR data show that MSN D2 knockout mice are more resilient to the sedative effects of alcohol, with only 50% of mice losing the righting reflex. Meanwhile, D2 autoreceptor knockout mice lose and regained the righting reflex after a high dose of alcohol similar to control mice. To further explore the rewarding and reinforcing aspects of alcohol in these transgenic mice, we examined intake parameters. MSN D2 knockout mice showed a higher preference for alcohol than controls in a two-bottle choice test and increased seeking in a self-administration paradigm. MSN D2 knockout mice show resilience to quinine adulteration of alcohol compared to controls. These results suggest that MSN striatal dopamine D2 receptors may be playing an important role in modulating the behavioral responses to alcohol. This may provide an explanation for the variation in individuals responses to the stimulant effects of alcohol and the resulting susceptibility to abuse and dependence. Project C: Lack of LRRK2, a Parkinsons disease-related protein, promotes compulsive-like and high alcohol intake in mice. High alcohol drinking and consumption despite aversive consequences is a characteristic of alcohol use disorders. The progression from controlled alcohol use to more compulsive drinking is influenced by many factors, such as psychological, environmental and genetic factors. In order to elucidate the genetic components implicated in the development of the compulsive alcohol consumption, we evaluated changes in the whole striatal transcriptome using a murine model of chronic and free choice alcohol intake. In this model, 60 outbred mice had access to alcohol in a three-bottle choice way during four stages: Acquisition (10 weeks), Withdraw (2 weeks), Re-exposure (2 weeks) and Adulteration (2 weeks, access to alcohol solutions adulterated with bitter tasting quinine). After treatment, animals were classified according to their individual alcohol intake as Light drinkers (preference for water throughout the experiment), Heavy drinkers (preference for alcohol, but significantly intake reduction after the alcohol taste-adulteration) and Inflexible drinkers (preference for alcohol even after the taste-adulteration). In the transcriptome analysis, we found that the expression of Lrrk2 gene was increased exclusively in those mice that were Inflexible drinkers. The Lrrk2 gene produces an AKAP protein which regulates the PKA availability in medium spiny neurons and it is responsible for controlling several neuronal functions. Further validations using the full knockout mice for the Lrrk2 gene (Lrrk2-KO) showed that those animals have an enhanced alcohol preference and consumption when compared with the wild types. Also, Lrrk2-KO mice achieved a higher breakpoint in a progressive ratio schedule, indicating an increased motivation to both alcohol seeking and taking. Compulsive-like behavior were accessed by measuring preference for 3 weeks and by alcohol adulteration test. Once again, Lrrk2-KO mice showed high alcohol intake despite the taste-adulteration, suggesting enhanced compulsive-like alcohol consumption in mice lacking the Lrrk2 gene. Furthermore, the Lrrk2-KO mice showed an increased basal anxiety-like behavior, spending a shorter time on the center of an open field arena. Taken together, these data support the hypothesis of the Lrrk2 gene contributes to the vulnerability to develop compulsive-like, out-of-controlled alcohol consumption and suggesting Lrrk2 gene could be an important target for treatment of alcohol use disorders. Project D: Corticotropin Releasing Factor (CRF) indirectly activates M5 receptors in the nucleus accumbens to facilitate appetitive behaviors. It was recently shown that CRF acting in the NAc potentiates dopamine release to promote approach behavior such as exploration of a novel object. Interestingly, repeated stress disrupts CRFs ability to increase dopamine and switches the behavioral response to CRF from approach to avoidance.We now show that CRF produces a robust concentration dependent increase in the firing of cholinergic interneurons (CIN) within the dorsal striatum and NAc. This increase CIN firing is via a CRF R1, PKA pathway dependent mechanism. Further, two components are driving CRF-mediated potentiation: one dependent and one independent of muscarinic receptors